1. Field of the Invention
The invention relates generally to a method for extracting antioxidants from and, more specifically, to an improved method of extracting antioxidants from species of the family Labiatae, in particular, rosemary (Rosemarinus officinalis), using tetrafluoroethane based solvent blends and which yields a liquid and oily extract that is readily mixed with an edible oil for addition to animal feeds and human food.
2. Background of the Prior Art
Worldwide demand for natural antioxidants has been rising due to safety concerns about synthetic food and feed additives and the public perception that natural food and feed supplements provide certain health benefits. The most important natural antioxidants being exploited commercially today are tocopherols. Tocopherols have a potent ability to inhibit lipid peroxidation in vivo by trapping peroxy radicals (Burton, G. W., and K. U Ingold (1989), in Vitamin E: Biochemistry and Health Implications, edited by A. T. Diplock, L. J. Machlin, L. Packer and W. A. Pryor, The New York Academy of Sciences, New York, pp 7-22). Various herbal extracts for use as natural antioxidants are being explored. Possibilities include the extraction of rosemary or other botanical sources. Such new antioxidants may play a role in combating carcinogenesis as well as the aging process, and may be applicable in the nutraceutical industry.
Among the various natural extracts available in the market are rosemary extracts, which are reported to be highly effective in retarding lipid oxidation and protecting living cells from the damaging oxidative stress (Chen, Q., H. Shi and C-T Ho (1992), xe2x80x9cEffects of rosemary extracts and major constituents on lipid oxidation and soybean lipoxygenase activityxe2x80x9d, J Am Oil Chem Soc 69: 999-1002; Wong, J. W., K. Hashimoto and T. Shibamoto (1995), xe2x80x9cAntioxidant activities of rosemary and sage extracts and vitamin E in a model meat systemxe2x80x9d, J Agric Food Chem 43: 2707-2712). These extracts are described as being superior to vitamin E, a well-known natural antioxidant and food supplement, in many food model systems (Lolinge, J, (1983), Natural antioxidants in Allen, J. C. and R. J. Hamilton eds, Rancidity in Foods, Elsevier Applied Science, London, Chapter 6). However, opposite findings are also documented. Wong et al. (1995) revealed that vitamin E is more effective than rosemary extract in a cooked beef homogenate. Additionally, rosemary extract is shown to be a synergist of vitamin E in stabilizing or retarding oxidation in sardine oil and fish muscle (Fang, X. and S. Wanda (1993), xe2x80x9cEnhancing the antioxidant effect of xcex1-tocopherol with rosemary extract in inhibiting catalyzed oxidation caused by Fe2+ and hemoproteinxe2x80x9d, Food Res Int 26: 405-411; Wanda, S. and X. Fang (1992), xe2x80x9cThe synergistic antioxidant effect of rosemary extract and xcex1-tocopherol in sardine oil model system and frozen-crushed fish meatxe2x80x9d, J Food Process Preserv 16: 263-274).
As to the extraction of rosemary, many authors report that polar solvents yield extracts with higher antioxidant activities (Chang, S. S., B. Ostric-Matijasevic, C-L Huang and OA-L Hsieh (1977), xe2x80x9cNatural antioxidants from rosemary and sagexe2x80x9d, J Food Sci 42: 1102-1106). Chen et al. (1992) found that hexane extracts of rosemary contained a higher content of carnosic acid and carnosol than methanol extracts do. Carnosic acid and carnosol are the effective antioxidant molecules in rosemary. Carnosic acid and carnosol have been suggested to account for over 90% of the antioxidant activity of rosemary extracts (Aruoma, O. I, B. Halliwell, R. Aeschbach and J. Loligers (1992) xe2x80x9cAntioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acidxe2x80x9d, Xenobiotica 22: 257-268). Antioxidant molecules in general, and rosemary antioxidants specifically, are by nature labile molecules especially when exposed to heat and/or air. During the harvest, the drying, and the regular solvent extraction of rosemary some oxidation is likely to occur. Through a process of chemical reactions, carnosic acid, the naturally occurring antioxidant molecule in rosemary, is believed to be the precursor to carnosol and many other antioxidants found therein (Wenkert, E., A. Fuchs, J. D. McChesney (1965), xe2x80x9cChemical artifacts from the family labiatexe2x80x9d, J. Org. Chem. 30: 2931-2934). It can be demonstrated that the freshly cut leaves of rosemary do not contain carnosol (Aeschbach, R. and L. Philippossian (1993), xe2x80x9cCarnosic acid obtention and usesxe2x80x9d, U.S. Pat. No. 5,256,700). Carnosic acid is about 10 times more effective as an antioxidant than carnosol (Aruoma et al., 1992) and it therefore is important for the high activity of a rosemary extract to minimize the damage to carnosic acid.
The antioxidant activity of commercially available rosemary products were compared with rosemary extracts prepared in the laboratory using various solvents for extraction. It was found that the antioxidant activity of commercial rosemary products was in the range of 2-5% when compared to mixed tocopherols. A methanol extract had 10% of the activity of mixed tocopherols. Methanol extraction, moreover, results in a dry powder that is difficult to dissolve into preferred carriers, such as edible oils. Accordingly, there was identified a goal to increase the specific activity of extracts of species of the family Labiate, including rosemary by optimizing the solvent extraction methodology and test alternate extraction technologies and to improve the handling characteristics of the extract.
The investigation into alternate extraction technology had two primary objectives. Firstly, to increase the specific activity of the rosemary extracts further for more efficient formulation into soybean oil or other carrier and, secondly, to identify technology allowing the removal of the essential oil fraction from the extracted material without oxidative destruction of the carnosic acid. One extraction technique investigated is based on tetrafluoroethane. Tetrafluoroethane has a boiling point of xe2x88x9227xc2x0 C. The technology utilizes the vapor pressure of the solvent at room temperature and allows extraction under mild conditions, therefore minimizing the oxidative decomposition of carnosic acid during the extraction process. Tetrafluoroethane is substantially apolar and is preferably blended with acetone in the extractions of rosemary described here.
A process for the extraction of antioxidants from rosemary preferably meets several criteria. It should be economical and also lead to a liquid or oil extract that can be formulated into a homogeneous, soybean oil-based final product that is largely free of odor. Methanol extracts the antioxidants from rosemary very effectively. However it leads to a dry powder extract and an inferior liquid final product after formulation into soybean oil. The extraction technology herein described is based on a solvent blend containing the solvent tetrafluoroethane (TFE) as major component. The optimum TFE-based solvent blend for the extraction of antioxidants from rosemary was identified and extraction parameters were defined. Among numerous solvent blends tested, an 80/15/5 blend of TFE/methanol/acetone, respectively, proved to be the most effective solvent resulting in a liquid extract with up to 35% of the tocopherol efficacy and an antioxidant yield of about 60% of the rosemary anti oxidants.
The advantages of TFE show that it is non-flammable, has a low boiling point, is environmentally acceptable (very low toxicity), and is easily handled. It has been found that at ambient or sub-ambient temperatures, TFE leaves behind the majority of the waxes and other non-fragrant materials normally extracted with conventional solvents (Wilde P. F., 1994. Fragrance Extraction. European Patent No. 0616821A1). Another advantage with the use of TFE is that no distillation must be employed due to its low boiling point.
A purpose of the invention is to identify a solvent blend and extraction parameters for the extraction of antioxidants of rosemary while attaining a high specific activity and retaining high extraction yields.
Another purpose of the present invention is to provide a method for extracting antioxidants from rosemary that yields a liquid oily extract that is readily mixed with a liquid product, such as soybean oil, for incorporation into animal feeds and human foods.